Current methods for early detection of breast cancer are not optimal. Mammography has a sensitivity of 75 percent - 90 percent the positive predictive value is low, approximately 25 percent. Furthermore, although resolution continues to improve, mammography is still dependent on the existence of a mass lesion. Because many breast tumors will already have metastasized by the time a mass is detectable, a significant portion of mammographically detected tumors in women undergoing regular screening will already be disseminated and incurable. Furthermore, mammography alone may not be sufficient for early detection in premenopausal women, particularly for young women with inherited susceptibility or other high risk profiles, for reasons related to breast density, tumor growth rates, and potential susceptibility to radiation-induced damage in women with germline predisposing mutations such as BRCA1/2. New methods for early detection that can serve as an adjunct to mammography are urgently needed. We are examining a novel approach that can be applied to early detection, based on comparing protein signatures in nipple aspirate fluid (NAF) and serum in women with and without breast cancer. This approach is based on the premise that women with early stages of neoplastic progression will have different patterns of protein expression that can be detected as secreted proteins shed into NAF or serum. In Years 1 and 2 we will first establish proof-of-principle by identifying breast cancer-specific protein signatures in fresh tumor tissue that contains invasive, in-situ and abnormal components. This will use a novel approach coupling Laser Capture Microscopy and Surface Enhanced Laser Desorption Ionization. After we have identified a consistent protein expression profile associated with malignancy we will evaluate the translational potential of this approach by determining whether these profiles can be distinguished in conveniently accessed body fluids, i.e. NAF and serum, from women with normal breasts, ductal carcinoma in-situ (DCIS), and early stage (stage I) invasive cancer. This will help to determine the stage of neoplastic progression at which early detection based on this approach is feasible. We have extensive data demonstrating the feasibility of this approach and our ability to enroll patients and obtain samples.